HGF is a unique cytokine having various activities not limited to controlling growth of hepatocytes, and growth of various epithelium cells, but also extended to enhancing motility of cells, and inducing morphogenesis with constructing three dimensional tissue. HGF takes a main role in generation or regeneration of tissues and organs as a mitogen, a motogen and a morphogen.
HGF is a heterodimeric protein comprising an .alpha. chain having a molecular weight of about 69 kD and a .beta. chain having a molecular weight of about 34 kD, and having a molecular weight 82-85 kD as a whole.
HGF acts as a growth factor for promoting growth of not only hepatocytes, but also renal tubule epithelial cells, keratinocytes, melanocytes, alveolar epithelial cells type II, gastric mucosal epithelial cells, vascular endthelial cells, and other epithelial cells.
HGF shows an activity for promoting growth of normal cells, but shows an activity for suppressing the proliferation of tumor cells (Tajima, H. et al., FEBS Lett., 291, 229, 1991).
HGF also shows an activity for enhancing motility of various epithelial cells such as MDCK cells (normal epithelial cells of renal tubule) and a motogen activity such as scattering colony of cultured said cells.
HGF is reported to have an activity of inducing morphogenesis of MDCK cells (Montesano, R. et al., Cell, 67, 901, 1991).
HGF inhibits leakage of a soluble enzyme from cultured hepatocytes prepared from an animal treated by carbon tetrachloride, a typical toxic substance to liver. These results show that HGF has an anti-hepatitis activity in vitro. HGF is also reported to have an anti-hepatitis activity in vivo (Takehara, T. et al., Biomed. Res., 12, 335, 1991).
HGF is also reported to enhance the activity of Na-K-ATPase in renal tubule epithelial cells and has been suggested to enhance renal functions (Ishibashi, T et al., Biochem. Biophys. Res. Commun., 182, 960, 1992).
Problems in administering HGF in vivo are fast clearance in vivo and that HGF originating from non-human species may have an antigenicity.
One of the effective ways to solve the problems is modifying the protein with a PEG reagent to delay the clearance and to reduce the antigenicity (Yoshimoto, T. et al., Jpn. J. Cancer Res., 77, 1264, 1986; Japanese patent application KOKAI 56-23587, 1981; Japanese patent application KOKAI 61-178926, 1986; Abuchowski, A. et al., Cancer Biochem. Biophys., 7, 175, 1984; Japanese patent application KOKAI 62-115280, 1987).
As described above, HGF has various biological activities and development of HGF derivatives as pharmaceutical agents is expected. It is considered important to develop a pharmaceutical composition for use in a method of treatment, comprising administration to a patient an effective amount of HGF for treating hepatic diseases, treating renal diseases, promoting growth epithelial cells, treating cancer, reducing side effects of anti-cancer reagents, treating lung diseases, treating gastric and duodenal diseases, treating cerebral and nerval injury, increasing platelets, treating hypoptoteinemia, healing wounds, increasing hemopoietic stem cells, restoring hair, and in skin cosmetics, but it is necessary to administer several ten .mu.g/kg to several mg/kg of HGF to have enough activities, and the administration amount may be relatively higher than other physiologically active protein. High dose may be disadvantageous because of unexpected side effects and high cost to manufacture a pharmaceutical product.
The most important problem to be solved is to improve the clearance in vivo, since it is reported that half life time of HGF in blood is quite short (.alpha. phase: about 2 minutes, .beta. phase: about 20 minutes). But a pharmaceutical composition for use in a method comprising administration of an effective amount of PEG-modified HGF having improved pharmacokinetics in vivo and improved activities to a patient for treating hepatic diseases, treating renal diseases, promoting growth epithelial cells, treating cancer, reducing side effects of anti-cancer reagents, treating lung diseases, treating gastric and duodenal diseases, treating cerebral and nerval injury, increasing platelets, treating hypoptoteinemia, healing wounds, increasing hemopoietic stem cells, restoring hair, and in skin cosmetics, has not been reported.
The subject of the present invention is to provide a PEG-modified HGF that can maintain bioactivities in vivo for a longer time to reduce the amount of administration, and has a specificity to targeting organ.